Stomatal CO 2 /bicarbonate sensor consists of two interacting protein kinases, Raf-like HT1 and non-kinase-activity requiring MPK12/MPK4.

The continuing rise in the atmospheric carbon dioxide (CO) concentration causes stomatal closing, thus critically affecting transpirational water loss, photosynthesis, and plant growth. However, the primary CO sensor remains unknown. Here, we show that elevated CO triggers interaction of the MAP kinases MPK4/MPK12 with the HT1 protein kinase, thus inhibiting HT1 kinase activity. At low CO, HT1 phosphorylates and activates the downstream negatively regulating CBC1 kinase. Physiologically relevant HT1-mediated phosphorylation sites in CBC1 are identified. In a genetic screen, we identify dominant active HT1 mutants that cause insensitivity to elevated CO. Dominant HT1 mutants abrogate the CO/bicarbonate-induced MPK4/12-HT1 interaction and HT1 inhibition, which may be explained by a structural AlphaFold2- and Gaussian-accelerated dynamics-generated model. Unexpectedly, MAP kinase activity is not required for CO sensor function and CO-triggered HT1 inhibition and stomatal closing. The presented findings reveal that MPK4/12 and HT1 together constitute the long-sought primary stomatal CO/bicarbonate sensor upstream of the CBC1 kinase in plants.